Production of xylanase by Aspergillus niger GIO and Bacillus megaterium through solid-state fermentation

Xylanase breaks xylan down to xylose, which is used in industries such as pulp and paper, food and feed, among others. The utilization of wastes for xylanase production is economical, hence this work aimed at producing xylanase through solid-state fermentation and characterizing the enzyme. Xylanase-producing strains of Bacillus megaterium and Aspergillus niger GIO were inoculated separately in a 5 and 10 day solid fermentation study on maize straw, rice straw, sawdust, corn cob, sugarcane bagasse, conifer litters, alkaline-pretreated maize straw (APM) and combined alkaline and biological-pretreated maize straw, respectively. The best substrate was selected for xylanase production. The crude enzyme was extracted from the fermentation medium and xylanase activity was characterized using parameters such as temperature, cations, pH and surfactants. Among different substrates, the highest xylanase activity of 3.18 U ml−1 was recorded when A. niger GIO was grown on APM. The xylanase produced by A. niger GIO and B. megaterium had the highest activities (3.67 U ml−1 and 3.36 U ml−1) at 40 °C after 30 and 45 min of incubation, respectively. Optimal xylanase activities (4.58 and 3.58 U ml−1) of A. niger GIO and B. megaterium , respectively, were observed at pH 5.0 and 6.2. All cations used enhanced xylanase activities except magnesium ion. Sodium dodecyl sulfate supported the highest xylanase activity of 6.13 and 6.90 U ml−1 for A. niger GIO and B. megaterium , respectively. High yields of xylanase were obtained from A. niger GIO and B. megaterium cultivated on APM. The xylanase activities were affected by pH, temperature, surfactants and cations.


INTRODUCTION
Lignocellulosic materials are abundant in nature and are principally made up of cellulose, hemicellulose and lignin [1,2]. Hemicellulose is the second most plentiful biological polymer in the world after cellulose and the main component of hemicellulose is xylan, which is broken down to xylose by the enzyme called xylanase [3]. Xylanase is a hydrolytic enzyme that breaks the β−1, 4-glycosidic bond of xylan in lignocellulolytic substrates to release xylose. Non-production of toxic materials is one of the advantages of using enzyme (xylanase) to break down xylan to xylose when compared with other methods of breaking down xylan [4][5][6] Xylanases have been applied in juice clarification [7], desizing yarn before weaving, deinking, biobleaching, improving the rheological property of bread, removal of waxy material from plant fibre, biorefinery [8], enhancement of plant immunity [9] and resistance to pathogens [9]. The use of xylan as a substrate for the production of xylanase has been discouraged because of the high cost of xylan [10]. Thus there is a need for alternative substrates that will contain xylan at a low cost. Lignocellulosic substrates such as agro-wastes can be used as alternatives to xylan in the production of xylanase because these substrates contain xylan and other nutrients necessary for the growth of micro-organisms [10,11]. Many micro-organisms such as Bacillus spp. [3,7,12,13], Aspergillus spp. [14][15][16], Penicillium spp. [10,17], Fusarium spp. [11,18], Streptomyces spp.

Statistical analysis
The obtained experimental data were analysed using analysis of variance (ANOVA) to determine the means with SPSS version 23 and the level of significance was set at P≤0.05. GraphPad Prism 6.0.1 (GraphPad Software, Inc., USA) was used for graphical presentation.

Screening for xylanase
The ability of A. niger GIO and B. megaterium to hydrolyse xylan on media supplemented with xylan is shown in Fig. 1a, b. A clear zone around the point of inoculation against the dark colour of undegraded xylan indicates that A. niger GIO (Fig. 1a) and B. megaterium (Fig. 1b) produced xylanase.

Production of xylanase
All substrates served as positive solid substrate production components for xylanase production. Fig. 2 shows the activities of xylanase produced on different agro-materials by A. niger GIO and B. megaterium. The xylanase activities of A. niger GIO and B. megaterium were significantly different (P≤0.05) from each other with combined pretreated maize straw, maize straw, rice straw and sawdust as substrates. The activities of xylanase produced by A. niger GIO on different agro-wastes as substrate ranged from 1.35 U ml −1 (sawdust) to 3.18 U ml −1 (APM). The highest activity of xylanase (3.18 U ml −1 ) produced by A. niger GIO using APM as a substrate was significantly different (P≤0.05) from those produced using other substrates. The highest activity of xylanase (3.15 U ml −1 ) produced by B. megaterium, was recorded when sugarcane baggase was used as the substrate. This was followed by the 3.14 U ml −1 activity realized with the use of sawdust. However, the least activity (1.93 U ml −1 ) was obtained when corn cob was the substrate for production. The activity of xylanase produced using APM by B. megaterium (3.11 U ml −1 ) was not significantly different (P>0.05) from the highest activity of xylanase (3.15 U ml −1 ) produced by the same organism using sugarcane baggase as substrate. APM substrate was selected for further studies.

Effect of temperature and incubation time
The effects of temperature and time on the activities of microbial xylanases are as shown in Table 1. The activities of xylanase produced by A. niger GIO and B. megaterium increased with an increase in temperature from 30 to 40 °C and decreased thereafter. At 30 °C, the activities of xylanase produced by A. niger GIO increased from 2.86 U ml −1 at 15 min to 3.31 U ml −1 at 30 min before a decrease in activities was recorded at 45 min. However, statistical analysis revealed that there was no significant difference (P>0.05) in activities recorded at 30, 45 and 60 min. The activities of xylanase produced by B. megaterium at 30 °C increased from 2.22 U ml −1 (15 min) to 3.13 U ml −1 (45 min), which then reduced to 2.88 U ml −1 after 60 min. The highest activities of xylanase produced by A. niger GIO (3.67 U ml −1 ) and B. megaterium (3.36 U ml −1 ) were recorded at a temperature of 40 °C at 30 and 45 min of incubation, respectively. At 40 °C, xylanase activities of B. megaterium at different incubation periods were not significantly different (P>0.05). A slight decrease in the activities of xylanase produced by A. niger GIO and B. megaterium were observed as the temperature increased to 50 °C, where the activities of xylanase produced by A. niger GIO and B. megaterium at different periods of incubation ranged from 3.28 to 3.41 U ml −1 and 3.01 to 3.21 U ml −1 , respectively, indicating a respective 7.1 and 4.5% reduction when compared to that at 40 °C. Statistical analysis revealed that there was no significant difference (P>0.05) in the activities of xylanase produced by A. niger GIO at different incubation periods. Further decrease in activities of xylanase produced by both A. niger GIO and B. megaterium was observed as the temperature of incubation increased to 60 °C. Compared to the peak activity recorded at 40 °C, at 60 °C the highest activity of xylanase produced by A. niger GIO (3.26 U ml −1 ) was recorded at 45 min of incubation, while that of B. megaterium (3.24 U ml −1 ) was recorded at 60 min of incubation, resulting in a respective 11.2 and 3.6% reduction in enzyme activity. Hence the xylanase produced by both micro-organisms was thermotolerant and thermostable at the temperatures tested. There was no significant difference (P>0.05) in the activities of xylanase produced by A. niger GIO at different times of incubation at 60 °C.  Fig. 3 shows the effect of pH on the activities of xylanase produced by A. niger GIO and B. megaterium. The activities of xylanase of A. niger GIO increased from 3.83 U ml −1 at pH 4.0 to 4.58 U ml −1 and thereafter decreased to 4.22 U ml −1 at pH 6.8. All activities of xylanase of A. niger GIO at all pH were not significantly different (P>0.05) from one another except the activity obtained at pH 4. Xylanase produced by B. megaterium had the highest activity (3.58 U ml −1 ) at pH 6.2. The activities of xylanase of B. megaterium recorded at pH 6.2 (3.58 U ml −1 ) and at pH 6.8 (3.53 U ml −1 ) were not significantly different (P>0.05).

Effect of metal ions and surfactants
Metal ions enhanced the activities of the xylanase produced by both A. niger GIO and B. megaterium (Fig. 4). With Ca 2+ , the highest activities of the A. niger GIO xylanase was 6.60 U ml −1 while that of B. megaterium was 5.40 U ml −1 . These values were significantly different (P≤0.05) from their respective controls (without metal ion). Activities of xylanase of A. niger GIO and B. megaterium were also improved with iron II ion, sodium ion, and potassium ion. However, the least activities were observed with magnesium ions for both xylanases of A. niger GIO (2.88 U ml −1 ) and B. megaterium (3.12 U ml −1 ). When compared to the controls, the magnesium ion-influenced xylanase from A. niger GIO and B. megaterium, respectively had 85.5 and 97.2% xylanase activity. The activities of xylanase of A. niger GIO and B. megaterium were not significantly different from their respective control (without metal ions) when magnesium and aluminium ions were used.
The effect of surfactants on the activities of microbial xylanase is shown in Table 2. The highest activities of xylanase of A. niger GIO (6.13 U ml −1 ) and B. megaterium (6.90 U ml −1 ) were positively influenced by sodium dodecyl sulfate and were significantly different from their respective control (without surfactant). The values of activity recorded with urea by A. niger GIO (5.51 U ml −1 ) and B. megaterium (4.20 U ml −1 ) were significantly higher than their respective controls. There was no activity of microbial xylanase recorded when EDTA was used as a surfactant.

DISCUSSION
Aspergillus and Bacillus species have been reported to produce a wide range of enzymes, especially lignocellulosic-degrading enzymes, which are involved in the breaking down of agricultural wastes [13,16,26]. A. niger GIO and B. megaterium used in this work hydrolysed xylan, with evidence of a clear zone on hyrolysed xylan media flooded with iodine against the dark colour of unhydrolysed xylan, as reported by Shakoori et al. [27].
Agro-wastes were used to produce xylanase in this study and the utilization of agro-wastes for the production of xylanase is a means of turning wastes into wealth. Many researchers have converted wastes to value-added products such as ethanol [28][29][30][31][32], with xylanase production from biomass residues as another example [10,[33][34][35]. Bastos et al. [11] used different agro-wastes (corn cob, barley bagasse, bacaba, rice husk, corn straw, pineapple crown, cassava husk and soybean husk) for the production of xylanase. Peach-palm waste was used for the production of xylanase by Carvalho et al. [22]. Zehra et al. [15] utilized banana peels for xylanase production. Utilization of wastes in the environment for the production of value-added products will create a safe environment and improve the economy.
The highest xylanase activities of A. niger GIO and B. megaterium were recorded at 40 °C in this study, which was similar to the work of Roy and Rowshanul [36], who reported the highest activities of xylanase being produced by a Bacillus species at 40 °C. Carvalho et al. [22] and Hernandez et al. [6] recorded the highest xylanase activity for their micro-organisms at 50 °C and 55 °C, respectively. The xylanase of A. niger GIO and B. megaterium in our work still retained 88.8 and 96.4% activity, respectively, at 60 °C when incubated for 45 and 15 min, respectively. This served as an indication of the thermostable properties of the xylanase produced. Generally, the optimal temperature for xylanase activity depends on the organisms producing it and xylanase activity is sensitive to temperature.
There was no significant difference in the xylanase activity of A. niger GIO from pH 4.6 to 6.8, which showed that the enzyme was relatively pH-stable and could be used effectively over an acidic to neutral pH range. Enzymes with relatively stable activities over pH ranges are important biotechnologically. The activities of xylanase are affected by pH because substrate bindings and catalysts depend on charge distributions of enzymes and substrates [37]. The optimal pH for the xylanase activities of A. niger GIO and B. megaterium were recorded at 5.0 and 6.2, respectively. Fungi have the ability to grow better than bacteria under an acidic environment and the activities of their enzymes are expected to be best at acidic ranges. It had been reported that acidic pH (3.0-5.5) favoured the activities of xylanase produced by fungi [10]. Bacteria grow better at a pH within the neutral region and their enzymes are expected to be active around this pH. Panthi et al. [37] reported an optimal pH of 6.0 for the xylanase activity of a Bacillus sp., while Hernandez et al. [6] recorded the highest xylanase activity for a bacterium at pH 6.5.
The addition of 5 mM potassium, sodium, calcium, iron (II) and aluminium metal ion improved the xylanase activities of A. niger GIO and B. megaterium, whereas magnesium ion repressed xylanase activities. Ferraz et al. [10] recorded an increase in xylanase activities when sodium, calcium and aluminium ions were added. The increase in xylanase activities observed  with some metal ions could be a result of the ability of the metal ion to stimulate the active site of enzymes [12]. The highest activity was recorded when calcium ion was used as a metal ion, which was similar to the report of Hernandez et al. [6]. Calcium ions are required structurally to maintain the active site of xylanase, while its absence will have negative effects on the recognition of substrate by the active site [6]. The reduction of xylanase activities upon the addition of magnesium ion corroborated the findings of Hernandez et al. [6], who also reported a reduction in xylanase activities upon the addition of magnesium as a metal ion. The decrease in enzyme activities by some metal ions might be due to the formation of insoluble complexes when the metal ions and xylan are mixed [37].
The addition of EDTA to the microbial xylanase had a negative impact on the activities of microbial enzyme in this research, and no activity was recorded in the presence of EDTA. The inhibitory effect of EDTA indicated that xylanases of A. niger GIO and B. megaterium are metallo enzymes. Metallo enzymes are inhibited by EDTA and EDTA could act as a chelator, trapping metals that are required for proper enzyme folding [6,13]. The xylanase activity of Bacillus subtilis JJBS250 was inhibited in the presence of EDTA [12]. Hernandez et al. [6] referred to EDTA as an organic acid inhibitor. Surfactants generally influence the function of proteins in cell signalling. Sodium dodecyl sulfate had a positive influence on the xylanase activities of the micro-organisms used in this work, but Gama et al. [19] and Sipriyadi et al. [20] reported that the addition of sodium dodecyl sulfate led to a decrease in the xylanase activities of different Streptomyces spp.
B. megaterium is a good producer of xylanase. Kareem et al. [38] used B. subtilis, B. megaterium, Bacillus cereus and Escherichia coli to produce xylanase and reported that B. megaterium was the best producer of xylanase among the different bacterial strains used in xylanase production. The xylanase activities of A. niger GIO (6.60 U ml −1 ) and B. megaterium (6.90 U ml −1 ) in this work were higher than the highest xylanase activity (3.6 U ml −1 ) recorded by Sipriyadi et al. [20], despite the different optimization steps the authors subjected the organism to.

CONCLUSION
A. niger GIO and B. megaterium utilized an array of agro-wastes as substrates for the production of xylanase. The highest activity of the xylanase produced by A. niger GIO and B. megaterium was recorded at 40 °C, while more than 88 % activity was still realized at 60 °C. The xylanases were active over the different pH ranges (pH 4.0 to 6.8). Potassium, sodium, calcium, iron II and aluminium ions significantly improved the activities of the xylanase produced by A. niger GIO and B. megaterium. Sodium dodecyl sulfate supported the best xylanase activities from both micro-organisms. The utilization of agro-wastes, which would otherwise have been a source of pollution in the environment, for xylanase production resulted in the conversion of wastes into wealth.

Funding information
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest
The authors declare that there are no conflicts of interest.

Peer review history
Comments: The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.  figure 3? All figure legends should provide enough information about the figure that a figure could be understood if the figure was removed from the manuscript and displayed alone. You need to add a lot more information into the legends at the moment without repeating too much of the main text. This is the same for tables. You need to define the elemental symbols in figure 4 Please rate the manuscript for methodological rigour Satisfactory

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data? Strongly support Significance needs to be shown on Figure 2 as well as in the text. Also, more analysis of the data is needed in the whole results section.
Significance has been shown More analysis has been added to results section It has been critically evaluated Line 280 -'some bacteria'. What are the species? Name them. Are they comparable to what you've done? Your statement is too generic.